Over the last quarter of a century antimicrobial drug resistance has become an increasingly serious problem in the clinic and the community. Fosfomycin is an inexpensive and potent broad-spectrum antibiotic effective against Gram-positive and Gram-negative bacteria. Shortly after its introduction into the clinic plasmid- mediated resistance to the drug was observed. Subsequently, research supported by this project discovered a large family of fosfomycin resistance proteins with three subfamilies now called FosA, FosB and FosX that catalyze the addition of glutathione, L-cysteine and water, respectively, to the antibiotic rendering it inactive. Plasmid encoded versions of FosA and FosX have been found in multi-drug resistant plasmids Serratia marcescens and P. putida. Perhaps more importantly, genomically encoded resistance proteins are extant in several select human pathogens, for example, (FosA) in Pseudomonas aeruginosa, (FosB) in Staphylococcus aureus and Bacillus anthracis, and (FosX) in Brucella melitensis, Listeria monocytogenes and Clostridium botulinum. The objectives of this project are to determine the structures and mechanisms of action of fosfomycin resistance proteins and to develop lead compounds for the effective inhibition of the enzymes as potential co-drugs in antimicrobial therapy. The research plan includes two specific aims. The first specific aim is to establish the evolutionary relationships among the FosA, FosB and FosX proteins. This will be accomplished by;(i) rational mutagenesis and directed evolution of FosA and FosX activities from a promiscuous progenitor encoded in the genome of Mesorhizobium loti and (ii) the solution of the three dimensional structure of at least one FosB protein. The second specific aim is to discover lead compounds for effective inhibitors of FosA, FosB and FosX. This aim will be addressed by (i) developing and validating high-throughput screening (HTS) assays for inhibitors of the three enzymes, (ii) screening the Vanderbilt Institute of Chemical Biology library of 160,000 compounds for inhibitors, (iii) follow-up of hits from the HTS with detailed inhibition studies and cell-based assays and, (iv) follow-up synthesis of compound libraries to be screened for more effective inhibitors. These investigations will establish the bases for the design of new drugs to counter resistance to fosfomycin that is encoded in the genomes of select human pathogens.